Caspase inhibitors and the use thereof

ABSTRACT

The present invention is directed to novel dipeptide thereof, represented by the general Formula I:where R1-R3, X and Y are defined herein. The present invention also relates to the discovery that compounds having Formula I are potent inhibitors of caspases and apoptotic cell death. Therefore, the inhibitors of this invention can retard or block cell death in a variety of clinical conditions in which the loss of cells, tissues or entire organs occurs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional application No.60/158,370, filed Oct. 12, 1999, abandoned, and U.S. Provisionalapplication No. 60/128,545, filed Apr. 9, 1999, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of medicinal chemistry. In particular,the invention relates to dipeptide caspase inhibitors with novelN-terminal blocking groups. The invention also relates to the use ofthese caspase inhibitors for reducing or treating apoptotic cell deathand/or reducing interleukin 1-β production.

2. Description of Background Art

Organisms eliminate unwanted cells by a process variously known asregulated cell death, programmed cell death or apoptosis. Such celldeath occurs as a normal aspect of animal development as well as intissue homeostasis and aging (Glucksmann, A., Biol. Rev. CambridgePhilos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie76:419-437 (1965); Ellis et al., Dev. 112:591-603 (1991); Vaux et al.,Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitatesmorphogenesis, removes harmful or otherwise abnormal cells andeliminates cells that have already performed their function.Additionally, apoptosis occurs in response to various physiologicalstresses, such as hypoxia or ischemia (PCT published applicationWO96/20721).

There are a number of morphological changes shared by cells experiencingregulated cell death, including plasma and nuclear membrane blebbing,cell shrinkage (condensation of nucleoplasm and cytoplasm), organellerelocalization and compaction, chromatin condensation and production ofapoptotic bodies (membrane enclosed particles containing intracellularmaterial) (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).

Apoptosis is achieved through an endogenous mechanism of cellularsuicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowenand Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activatesits internally encoded suicide program as a result of either internal orexternal signals. The suicide program is executed through the activationof a carefully regulated genetic program (Wylie et al., Int. Rev. Cyt.68:251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7:663 (1991)).Apoptotic cells and bodies are usually recognized and cleared byneighboring cells or macrophages before lysis. Because of this clearancemechanism, inflammation is not induced despite the clearance of greatnumbers of cells (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

Mammalian interleukin-1β (IL-1β) plays an important role in variouspathologic processes, including chronic and acute inflammation andautoimmune diseases (Oppenheim et. al. Immunology Today, 7:45-56(1986)). IL-1β is synthesized as a cell associated precursor polypeptide(pro-IL-1β) that is unable to bind IL-1 receptors and is biologicallyinactive (Mosley et al., J. Biol. Chem. 262:2941-2944 (1987)). Byinhibiting conversion of precursor IL-1β to mature IL-1β, the activityof interleukin-1 can be inhibited. Interleukin-1β converting enzyme(ICE) is a protease responsible for the activation of interleukin-1β(IL-1β) (Thornberry et al., Nature 356:768 (1992); Yuan et al., Cell75:641 (1993)). ICE is a substrate-specific cysteine protease thatcleaves the inactive prointerleukin-1 to produce the mature IL-1. Thegenes that encode for ICE and CPP32 are members of the mammalianICE/Ced-3 family of genes which presently includes at least twelvemembers: ICE, CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2,MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICE_(re1)III. The proteolyticactivity of this family of cysteine proteases, whose active site (acysteine residue) is essential for ICE-mediated apoptosis, appearscritical in mediating cell death (Miura et al., Cell 75:653-660 (1993)).This gene family has recently been named caspases (Alnernri et. al.Cell, 87:171 (1996), and Thornberry et. al., J. Biol. Chem.272:17907-17911 (1997)) and divided into three groups according to itsknown functions. Table 1 summarizes these known caspases.

TABLE I Enzyme* Group I: mediators of inflammation Casnase-1 (ICE)Casnase-4 (ICE_(rel)-II, TX, ICH-2) Casnase-5 (ICE_(rel)-III, TY) GroupII: effectors of apoptosis Casnase-2 (ICH-1, mNEDD2) Casnase-3 (apopain,CPP-32, YAMA) Casnase-7 (Mch-3, ICE-LAP3, CMH-1) Group III: activatorsof apoptosis Casnase-6 (Mch2) Casnase-8 (MACH, ELICE, Mch5) Casnase-9(ICE-LAP6, Mch6) Casnase-10

IL-1 is also a cytokine involved in mediating a wide range of biologicalresponses including inflammation, septic shock, wound healing,hematopoiesis and growth of certain leukemias (Dinarello, C. A., Blood77:1627-1652 (1991); diGiovine et al., Immunology Today 11:13 (1990)).

Many potent caspase inhibitors have been prepared based on the peptidesubstrate structures of caspases. However, in contrast to their potencyin vitro, not too many inhibitors with good efficacy (IC₅₀<1 μM) inwhole-cell models of apoptosis have been reported (Thornberry, N. A.Chem. Biol. 5:R97-103 (1998)). Therefore the need exists for cell deathinhibitors that are efficacy in whole-cell models of apoptosis andactive in animal model of apoptosis. These inhibitors thus can beemployed as therapeutic agents to treat disease states in whichregulated cell death and the cytokine activity of IL-1 play a role.

WO 93/05071 discloses peptide ICE inhibitors with the formula:

 Z—Q₂—Asp—Q₁

wherein Z is an N-terminal protecting group; Q₂ is 0 to 4 amino acidssuch that the sequence Q₂-Asp corresponds to at least a portion of thesequence Ala-Tyr-Val-His-Asp (SEQ ID NO:1); Q₁ comprises anelectronegative leaving group.

WO 96/03982 discloses aspartic acid analogs as ICE inhibitors with theformula:

wherein R₂ is H or alkyl; R₃ is a leaving group such as halogen; R₁ isheteroaryl-CO or an amino acid residue.

U.S. Pat. No. 5,585,357 discloses peptidic ketones as ICE inhibitorswith the formula:

wherein n is 0-2; each AA is independently L-valine or L-alanine; R₁ isselected from the group consisting of N-benzyloxycarbonyl and othergroups; R₈, R₉, R₁₀ are each independently hydrogen, lower alkyl andother groups.

Mjalli et al. (Bioorg. Med. Chem. Lett. 3:2689-2692 (1993)) report thepreparation of peptide phenylalkyl ketones as reversible inhibitors ofICE, such as:

Thornberry et al. (Biochemistry 33:3934-3940 (1994)) report theirreversible inactivation of ICE by peptide acyloxymethyl ketones:

wherein Ar is COPh-2,6-(CF₃)₂, COPh-2,6-(CH₃)₂, Ph-F₅ and other groups.

Dolle et al. (J. Med. Chem. 37:563-564 (1994)) report the preparation ofP₁ aspartate-based peptide α-((2,6-dichlorobenzoyl)oxy)methyl ketones aspotent time-dependent inhibitors of ICE, such as:

Mjalli et al. (Bioorg. Med. Chem. Lett. 4:1965-1968, (1994)) report thepreparation of activated ketones as potent reversible inhibitors of ICE:

wherein X is NH(CH₂)₂, OCO(CH₂)₂, S(CH₂)₃ and other groups.

Dolle et al. (J. Med. Chem. 37:3863-3866 (1994)) report the preparationof α-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methyl ketones asirreversible inhibitor of ICE, such as:

Mjalli et al. (Bioorg. Med. Chem. Lett. 5:1405-1408 (1995)) reportinhibition of ICE by N-acyl-Aspartic acid ketones:

wherein XR₂ is NH(CH₂)₂Ph, OCO(CH₂)₂cyclohexyl and other groups.

Mjalli et al. (Bioorg. Med. Chem. Lett. 5:1409-1414 (1995)) reportinhibition of ICE by N-acyl-aspartyl aryloxymethyl ketones, such as:

Dolle et al. (J. Med. Chem. 38:220-222 (1995)) report the preparation ofaspartyl α-((diphenylphosphinyl)oxy)methyl ketones as irreversibleinhibitors of ICE, such as:

Graybill et al. (Bioorg. Med. Chem. Lett. 7:41-46 (1997)) report thepreparation of α-((tetronoyl)oxy)- and α-((tetramoyl)oxy)methyl ketonesas inhibitors of ICE, such as:

Semple et al. (Bioorg. Med. Chem. Lett. 8:959-964 (1998)) report thepreparation of peptidomimetic aminomethylene ketones as inhibitors ofICE, such as:

Okamoto et al. (Chem. Pharm. Bull. 47:11-21 (1999)) report thepreparation of peptide based ICE inhibitors with the P1 carboxyl groupconverted to an amide, such as:

EP618223 patent application discloses inhibitors of ICE asanti-inflammatory agents:

R—A₁—A₂—X—A₃

wherein R is a protecting group or optionally substituted benzyloxy; A₁is an α-hydroxy or α-amino acid residue or a radical of formula:

wherein ring A is optionally substituted by hydroxy or C₁₋₄ alkoxy andR_(a) is CO or CS; A₂ is an α-hydroxy or α-amino acid residue or A₁ andA₂ form together a pseudo-dipeptide or a dipeptide mimetic residue; X isa residue derived from Asp; A₃ is —CH₂—X₁—CO—Y₁, —CH₂—O—Y₂, —CH₂—S—Y₃,wherein X₁ is O or S; Y₁, Y₂ or Y₃ is cycloaliphatic residue, andoptionally substituted aryl.

WO99/18781 discloses dipeptides of formula I:

wherein R₁ is an N-terminal protecting group; AA is a residue of anynatural or non-natural α-amino acid, β-amino acid, derivatives of anα-amino acid or β-amino acid; R₂ is H or CH₂R₄ where R₄ is anelectronegative leaving group, and R₃ is alkyl or H, provided that AA isnot His, Tyr, Pro or Phe. These dipeptides are surprisingly potentcaspase inhibitors of apoptosis in cell based systems. These compoundsare systemically active in vivo and are potent inhibitors ofantiFas-induced lethality in a mouse liver apoptosis model and haverobust neuroprotective effects in a rat model of ischemic stroke.

WO 99/47154 disclose dipeptides of formula I:

wherein R₁ is a N-terminal protecting group; AA is a residue of anon-natural α-amino acid or β-amino acid; R₂ is an optionallysubstituted alkyl or H.

WO 00/01666 disclosed c-terminal modified oxamyl dipeptides asinhibitors of the ICE/ced-3 family of cysteine proteases:

wherein A is a natural or unnatural amino acid; B is a hydrogen atom, adeuterium atom, alkyl, cycloalkyl and other groups; R₁ is alkyl,cycloalkyl and other groups, R₂ is hydrogen, lower alkyl and othergroups.

SUMMARY OF THE INVENTION

The invention relates to compound of Formula I:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

R₁ is an optionally substituted alkyl or hydrogen;

R₂ is hydrogen or optionally substituted alkyl;

R₃ is an alkyl, saturated carbocyclic, partially saturated carbocyclic,aryl, saturated heteroclyclic, partially saturated heterocyclic orheteroaryl group, wherein said group is optionally substituted;

X is O, S, NR₄, or (CR₄R₅)_(n), where R₄ and R₅ are, at each occurrence,independently selected from the group consisting of hydrogen, alkyl andcycloalkyl, and n is 0, 1, 2, or 3; or

X is NR₄, and R₃ and R₄ are taken together with the nitrogen atom towhich they are attached to form a saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; or

X is CR₄R₅, and R₃ and R₄ are taken together with the carbon atom towhich they are attached to form a saturated carbocyclic, partiallysaturated carbocyclic, aryl, saturated heterocyclic, partially saturatedheterocyclic or oxygen-containing heteroaryl group, wherein said groupis optionally substituted; and

Y is a residue of a natural or non-natural amino acid;

provided that when X is O, then R₃ is not unsubstituted benzyl ort-butyl; and when X is CH₂, then R₃ is not hydrogen.

The invention relates to the discovery that the compounds represented byFormula I are inhibitors of caspases. The invention also relates to theuse of the compounds of the invention for reducing, preventing ortreating maladies in which apoptotic cell death is either a causativefactor or a result. Examples of uses for the present invention includeprotecting the nervous system following focal ischemia and globalischemia; treating neurodegenerative disorders such as Alzheimer'sdisease, Huntington's Disease, prion diseases, Parkinson's Disease,multiple sclerosis, amyotrophic lateral sclerosis, ataxia,telangiectasia, and spinobulbar atrophy; treating heart diseaseincluding myocardial infarction, congestive heart failure andcardiomyopathy; treating retinal disorders; treating autoimmunedisorders including lupus erythematosus, rheumatoid arthritis, type Idiabetes, Sjögren's syndrome and glomerulonephritis; treating polycystickidney disease and anemia/erythropoiesis; treating immune systemdisorders, including AIDS and SCIDS; treating or ameliorating sepsis,reducing or preventing cell, tissue and organ damage duringtransplantation; reducing or preventing cell line death in industrialbiotechnology; reducing or preventing alopecia (hair loss); and reducingthe premature death of skin cells.

The present invention provides pharmaceutical compositions comprising acompound of Formula I in an effective amount to reduce apoptotic celldeath in an animal.

The present invention also provides preservation or storage solutionsfor mammalian organs or tissue, or growth media for mammalian or yeastcells, wherein an effective amount of a compound of Formula I isincluded in said solutions or media in order to reduce apoptotic celldeath in said organs, tissue or cells.

The invention also relates to the use of caspase inhibitors fortreating, ameliorating, and preventing non-cancer cell death duringchemotherapy and radiation therapy and for treating and ameliorating theside effects of chemotherapy and radiation therapy of cancer.

In particular, the invention relates to a method of treating,ameliorating or preventing oral mucositis, gastrointestinal mucositis,bladder mucositis, proctitis, bone marrow cell death, skin cell deathand hair loss resulting from chemotherapy or radiation therapy of cancerin an animal, comprising administering to the animal in need thereof aneffective amount of a caspase inhibitor.

DETAILED DESCRIPTION OF THE INVENTION

The inhibitors of caspases and apoptotic cell death of the presentinvention are compounds having the general Formula I:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

R₁ is an optionally substituted alkyl or hydrogen;

R₂ is hydrogen or optionally substituted alkyl;

R₃ is an alkyl, saturated carbocyclic, partially saturated carbocyclic,aryl, saturated heterocyclic, partially saturated heterocyclic orheteroaryl group, wherein said group is optionally substituted;

X is O, S, NR₄, or (CR₄R₅)_(n), where R₄ and R₅ are, at each occurrence,independently selected from the group consisting of hydrogen, alkyl andcycloalkyl, and n is 0, 1, 2, or 3; or

X is NR_(4,) and R₃ and R₄ are taken together with the nitrogen atom towhich they are attached to form a saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; or

X is CR₄R₅, and R₃ and R₄ are taken together with the carbon atom towhich they are attached to form a saturated carbocyclic, partiallysaturated carbocyclic, aryl, saturated heterocyclic, partially saturatedheterocyclic or oxygen-containing heteroaryl group, wherein said groupis optionally substituted; and

Y is a residue of a natural or non-natural amino acid;

provided that when X is O, then R₃ is not unsubstituted benzyl ort-butyl; and when X is CH₂, then R₃ is not hydrogen.

With respect to R₁, preferred alkyl groups are C₁₋₆ alkyl groups, e.g.,methyl, ethyl, propyl, isopropyl, isobutyl, pentyl and hexyl groups; andsubstituted C₁₋₆ alkyl groups, e.g., CH₂OCH₃ and CH₂OCOCH₃ (AM oracetoxymethyl).

Preferred R₂ are alkyl group substituted by electronegative group orleaving group, including fluoromethyl, chloromethyl, alkoxymethyl,aryloxymethyl, alkylthiomethyl, arylthiomethyl, aminomethyl,acyloxymethyl, and arylacyloxymethyl. Other examples of optionalsubstituents that may be present at the R₂ alkyl group include, withoutlimitation, 3-pyrazolyloxy optionally substituted at the 2, 4 and5-positions with lower alkyl;3-(1-phenyl-3-trifluoromethyl)pyrazolyloxy;2,6-di(trifluoromethyl)benzoyloxy; 2,6-dimethylbenzoyloxy;pentafluoro-phenoxy; tetrafluorophenoxy; 2,6-dichlorobenzoyloxy;2-(3-(2-imidazolyl)naphthyl)oxy; diphenylphosphinyloxy; tetronyloxy; andtetramoyloxy.

The R₃ group in compounds of Formula I is designed to function as the P₃side chain in a tripeptide. Structure A is an example of a dipeptideinhibitor of Formula I. In comparison, structure B is an example of atripeptide inhibitor.

Preferred X is O, NH and CH₂. With respect to R₃, preferred alkyl aremethyl, ethyl, isopropyl, isobutyl; preferred substituents on alkyl arehydroxy, carboxy, halogen, C₄-C₇ cycloalkyl, saturated and partiallysaturated heterocyclic, aryl or heteroaryl; preferred cycloalkyl arecyclopentyl and cyclohexyl; preferred saturated and partially saturatedheterocyclic groups are piperidinyl and morpholinyl; preferred aryls arephenyl and naphthyl; preferred heteroaryls are pyridyl, indolyl, furyland thienyl; preferred substituents in the aryl and heteroaryl aremethyl, ethyl, chloro, fluoro, bromo, trifluoromethyl, methoxy, hydroxy,carboxy, cyano and nitro.

With respect to Y, preferred natural and non-natural amino acid arevaline, isoleucine, leucine, proline, alanine, phenylalanine,methionine, serine, threonine, tryptophan, tyrosine, 2-aminobutyricacid, cyclohexylglycine, phenylglycine, cyclopentylglycine andt-butylglycine. Especially preferred amino acids are valine, isoleucine,leucine, alanine, phenylalanine, cyclohexylalanine, 2-aminobutyric acid,cyclohexylglycine, and phenylglycine.

The invention relates to the discovery that the compounds represented byFormula I are inhibitors of caspases. These inhibitors slow or blockcell death in a variety of clinical conditions and industrialapplications in which the loss of cells, tissues or entire organsoccurs. Therefore, the invention is also related to methods of treating,preventing or reducing conditions in which apoptosis plays a role. Theseconditions are more fully described below.

The methods comprise administering to an animal in need of suchtreatment an inhibitor of the present invention, or a pharmaceuticallyacceptable salt or prodrug thereof, in an amount effective to inhibitapoptotic cell death.

Another group of preferred embodiments of the present invention that maybe employed as inhibitors of caspases are represented by Formula II:

or pharmaceutically acceptable salts or prodrugs thereof wherein R₁, R₂,X and

Y are as defined previously with respect to Formula I; and

A is CR₆ or nitrogen;

B is CR₇ or nitrogen;

C is CR₈ or nitrogen;

D is CR₉ or nitrogen;

E is CR₁₀ or nitrogen; provided that not more than three of A, B, C, Dand E are nitrogen; and R₆-R₁₀ independently are hydrogen, halo, C₁-C₆haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₀ ary(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or

one of R₆ and R₇, or R₇ and R₈, or R₈ and R₉, or R₉ and R₁₀ are takentogether with the carbon atoms to which they are attached to form acarbocycle or heterocycle.

Examples of bridges formed by R₆ and R₇, or R₇ and R₈, or R₈ and R₉, orR₉ and R₁₀ taken together are —OCH₂O—, —OCF₂O—, —(CH₂)₃—, —(CH₂)₄—,—OCH₂CH₂O—, —CH₂N(R₁₃)CH₂—, —CH₂CH₂N(R₁₃)CH₂—, —CH₂N(R₁₃)CH₂CH₂—,—N(R₁₃)—CH═CH—, —CH═CH—N(R₁₃)—, —O—CH═CH—, —CH═CH—O—, —S—CH═CH—,—CH═CH—S—, —N═CH—CH═CH—, —CH═N—CH═CH—, —CH═CH—N═CH—, —CH═CH—CH═N—,—N═CH—CH═N—, and —CH═CH—CH═CH—;

where R₁₃ is hydrogen, alkyl or cycloalkyl;

provided that when X is O, A is CR₆, B is CR₇, C is CR₈, D is CR₉ and Eis CR₁₀, then at least one of the R₆-R₁₀ is not a hydrogen.

Preferred R₁ is H, Me, Et, t-Bu or AM. Preferred R₂ is fluoromethyl,acyloxymethyl, arylacyloxymethyl, aryloxymethyl, phophinyloxymethyl oraminomethyl.

Another preferred group of the inhibitors of caspases and apoptotic celldeath of the present invention are compounds having the general FormulaIII:

or pharmaceutically acceptable salts or prodrugs thereof wherein R₁, R₂,R₃ and Y are as defined previously with respect to Formula I.

Preferred R₁ is H, Me, Et, t-Bu or AM. Preferred R₂ is fluoromethyl,acyloxymethyl, arylacyloxymethyl, aryloxymethyl, phophinyloxymethyl oraminomethyl. Preferred R₃ is optionally substituted alkyl or aryl.Preferred Y is valine, isoleucine, leucine, alanine, phenylalanine,cyclohexylalanine, 2-aminobutyric acid, cyclohexylglycine orphenylglycine.

Exemplary preferred inhibitors of caspases having Formular I include,without limitation:

2-Chlorobenzyloxycarbonyl-Val-Asp-fmk,

3-Chlorobenzyloxycarbonyl-Val-Asp-fmk,

4-Chlorobenzyloxycarbonyl-Val-Asp-fmk,

Phenethoxycarbonyl-Val-Asp-fmk,

Cyclohexylmethoxycarbonyl-Val-Asp-fmk,

Methoxycarbonyl-Val-Asp-fmk,

Ethoxycarbonyl-Val-Asp-fmk,

Isopropyloxycarbonyl-Val-Asp-fmk,

2-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,

3-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,

4-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,

Phenylacetyl-Val-Asp-fmk,

4-Nitrobenzyloxycarbonyl-Val-Asp-fmk,

2,5-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,

3,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,

3,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,

2,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,

2,6-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,

2,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,

2,4-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,

4-Ethylbenzyloxycarbonyl-Val-Asp-fmk,

4-Bromobenzyloxycarbonyl-Val-Asp-fmk,

4-Fluorobenzyloxycarbonyl-Val-Asp-fmk,

Cyclopentylmethoxycarbonyl-Val-Asp-fmk,

4-Trifluoromethylbenzyloxycarbonyl-Val-Asp-fmk,

3-Phenylpropionyl-Val-Asp-fmk,

Benzylaminocarbonyl-Val-Asp-fmk,

3-Phenylpropyloxycarbonyl-Val-Asp-fmk,

2,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,

3,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,

4-Morpholinecarbonyl-Val-Asp-fmk,

4-Pyridylmethoxycarbonyl-Val-Asp-fmk,

2-Pyridylmethoxycarbonyl-Val-Asp-fmk,

2,6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone,

Isobutoxycarbonyl-Val-Asp-fmk,

Propionyl-Val-Asp-fmk,

Benzyl-glutaryl-Val-Asp-fmk,Glutaryl-Val-Asp-fmk,

3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk,

3-(5-Bromo-2-hydroxyphenyl)propionyl-Val-Asp-fmk,

3-Fluorobenzyloxycarbonyl-Val-Asp-fmk,

2-Fluorobenzyloxycarbonyl-Val-Asp-fmk,

3-Methylbenzyloxycarbonyl-Val-Asp-fmk,

2-Chloro-4-fluorobenzyloxycarbonyl-Val-Asp-fmk,

2-Naphthylmethoxycarbonyl-Val-Asp-fmk,

p-Toluenesulfonyl-Val-Asp-fmk, and

p-Toluenesulfonyl-Phe-Asp-fmk.

where fmk is fluoromethylketone and DCB is 2,6-dichlorobenzoyloxy.

Useful aryl groups are C₆₋₁₄ aryl, especially C₆₋₁₀ aryl. Typical C₆₋₁₄aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl,indenyl, azulenyl, biphenyl, biphenylenyl nylenyl and fluorenyl groups.

Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

Useful saturated or partially saturated carbocyclic groups arecycloalkyl groups as defined above, as well as cycloalkenyl groups, suchas cyclopentenyl, cycloheptenyl and cyclooctenyl.

Useful halo or halogen groups include fluorine, chlorine, bromine andiodine.

Useful alkyl groups include straight-chained and branched C₁₋₁₀ alkylgroups, more preferably C₁₋₆ alkyl groups. Typical C₁₋₁₀ alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,3-pentyl, hexyl and octyl groups. Also contemplated is a trimethylenegroup substituted on two adjoining positions on the benzene ring of thecompounds of the invention.

Useful arylalkyl groups include any of the above-mentioned C₁₋₁₀ alkylgroups substituted by any of the above-mentioned C₆ ₆₋₁₄ aryl groups.Useful values include benzyl, phenethyl and naphthylmethyl.

Useful haloalkyl groups include C₁₋₁₀ alkyl groups substituted by one ormore fluorine, chlorine, bromine or iodine atoms, e.g., fluoromethyl,difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl,chloromethyl, chlorofluoromethyl and trichloromethyl groups.

Useful alkoxy groups include oxygen substituted by one of the C₁₋₁₀alkyl groups mentioned above.

Useful alkylthio groups include sulphur substituted by one of the C₁₋₁₀alkyl groups mentioned above. Also included are the sulfoxides andsulfones of such alkylthio groups.

Useful acylamino groups are any C₁₋₆ acyl (alkanoyl) attached to anamino nitrogen, e.g., acetamido, propionamido, butanoylamido,pentanoylamido, hexanoylamido as well as aryl-substituted C₂₋₆substituted acyl groups.

Useful acyloxy groups are any C₁₋₆ acyl (alkanoyl) attached to an oxy(—O—) group, e.g., formyloxy, acetoxy, propionoyloxy, butanoyloxy,pentanoyloxy, hexanoyloxy and the like.

Useful arylacyloxy groups include any of the aryl groups mentioned abovesubstituted on any of the acyloxy groups mentioned above, e.g.,2,6-dichlorobenzoyloxy, 2,6-difluorobenzoyloxy and2,6-di-(trifluoromethyl)-benzoyloxy groups.

Useful amino groups include —NH₂, —NHR₁₁, and —NR₁₁R₁₂, wherein R₁₁ andR₁₂ are C₁₋₁₀ alkyl or cycloalkyl groups as defined above.

Useful saturated or partially saturated heterocyclic groups includetetrahydrofuranyl, pyranyl, piperidinyl, piperizinyl, pyrrolidinyl,imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl,morpholinyl, isochromanyl, chromanyl, pyrazolidinyl pyrazolinyl,tetronoyl and tetramoyl groups.

Useful heteroaryl groups include any one of the following: thienyl,benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, chromenyl, coumarinyl, xanthenyl, phenoxanthiinyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl,phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl and 2-oxobenzimidazolyl. Where the heteroaryl group containsa nitrogen atom in a ring, such nitrogen atom may be in the form of anN-oxide, e.g., a pyridyl N-oxide, pyrazinyl N-oxide, pyrimidinyl N-oxideand the like.

Optional substituents include one or more alkyl; halo; haloalkyl;cycloalkyl; hydroxy; carboxy; phosphinyloxy; aryl optionally substitutedwith one or more lower alkyl, halo, amino, alkylamino, dialkylamino,alkoxy, hydroxy, carboxy, haloalkyl or heteroaryl groups; aryloxyoptionally substituted with one or more lower alkyl, halo, amino,alkylamino, dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl orheteroaryl groups; aralkyl; heteroaryl optionally substituted with oneor more lower alkyl, halo, amino, alkylamino, dialkylamino, alkoxy,hydroxy, carboxy, haloalkyl and aryl groups; heteroaryloxy optionallysubstituted with one or more lower alkyl, halo, amino, alkylamino,dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl and aryl groups;alkoxy; alkylthio; arylthio; amino; alkylamino; dialkylamino; acyloxy;arylacyloxy optionally substituted with one or more lower alkyl, halo,amino, alkylamino, dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl andaryl groups; diphenylphosphinyloxy optionally substituted with one ormore lower alkyl, halo, amino, alkylamino, dialkylamino, alkoxy,hydroxy, carboxy, or haloalkyl groups; heterocyclo optionallysubstituted with one or more lower alkyl, halo, amino, alkylamino,dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl and aryl groups;heterocycloalkyloxy optionally substituted with one or more lower alkyl,halo, amino, alkylamino, dialkylamino, alkoxy, hydroxy, carboxy,haloalkyl and aryl groups; partially saturated heterocycloalkyloptionally substituted with one or more lower alkyl, halo, amino,alkylamino, dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl and arylgroups; or partially saturated heterocycloalkyloxy optionallysubstituted with one or more lower alkyl, halo, amino, alkylamino,dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl and aryl groups.

Certain of the compounds of the present invention may exist asstereoisomers including optical isomers. The invention includes allstereoisomers and both the racemic mixtures of such stereoisomers aswell as the individual enantiomers that may be separated according tomethods that are well known to those of ordinary skill in the art.

Examples of pharmaceutically acceptable addition salts include inorganicand organic acid addition salts such as hydrochloride, hydrobromide,phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate,mandelate and oxalate; and inorganic and organic base addition saltswith bases such as sodium hydroxy and Tris(hydroxymethyl)aminomethane(TRIS, tromethane).

Examples of prodrugs include compounds of Formula I wherein R₁ is analkyl group or substituted alkyl group such as CH₂OCH₃ and CH₂OCOCH₃ (AMester).

The invention is also directed to a method for treating disordersresponsive to the inhibition of caspases in animals suffering thereof.Particular preferred embodiments of compounds for use in the method ofthis invention are represented by previously defined Formula I.

The compounds of this invention may be prepared using methods known tothose skilled in the art. Specifically, compounds with Formula I can beprepared as illustrated by exemplary reactions in Scheme 1. Theintermediate 1 was prepared according to Revesz et al. (TetrahedronLett. 35:9693-9696 (1994)). Coupling of 1 with a N-protected amino acid,such as (2-chlorobenzyloxycarbonyl)-Val, which was prepared from2-chlorobenzyl chloroformate and Valine, gave amide 2. Oxidation of 2 byDess-Martin reagent according to Revesz et al. (Tetrahedron Lett.35:9693-9696 (1994)) gave 3 as a mixture of diastereomers. The oxidationalso can be done using other agents such as pyridinium chlorochromate(PCC) or pyridinium dichromate (PDC). Acid catalyzed cleavage of theester gave the free acid 4.

Other N-protected amino acid can be prepared as illustrated by exemplaryreactions in Scheme 2-4.

Compounds of Formula III with substituted-sulfonyl as N-protecting groupcan be prepared similar to what described in Scheme 1. Examples ofsulfonyl protected amino acids which can be used for the preparation ofnovel caspase inhibitors of Formula III are shown in Scheme 5.

Other N-protecting group with special function also can be used. Forinstance, an antioxidant such as Trolox can be introduced as theprotecting group. The compound can be prepared as shown in Scheme 6.Alternatively, the compound can be prepared as shown in Scheme 7. Thecompound will combine the property of a caspase inhibitor with anantioxidant, which might be more efficacious as a neuroprotectant forthe treatment of stroke (Pierre-Etienne Chabrier et al., PNAS96:10824-10829 (1999)).

A fluorescent dye also can be introduced as the protecting group, suchas the compounds shown in Scheme 8.

These compounds can inhibit caspase and resulted in the attachment ofthe fluorescent dye to the caspase. Therefore these molecules should beuseful for the labeling of caspase and detection of caspase activity inthe cells. These compounds can be prepared as illustrated by exemplaryreactions in Scheme 9.

Preferred fluorescent protecting groups of formula R₃—X—C(O)— include,for example:

An important aspect of the present invention is the discovery thatcompounds having Formula I are inhibitors of caspases. Therefore, theseinhibitors are expected to slow or block cell death in a variety ofclinical conditions in which the loss of cells, tissues or entire organsoccurs.

The cell death inhibitors of the present invention can be used to reduceor prevent cell death in the nervous system (brain, spinal cord, andperipheral nervous system) under various conditions of ischemia andexcitotoxicity, including, but not limited to, focal ischemia due tostroke and global ischemia due to cardiac arrest, as well as spinal cordinjury (Emery et al., J. Neurosurgery 89:911-920 (1998)). One particularusage is to treat the effects of oxygen deprivation which can occurduring the birth of infants in high-risk labors or drowning. The celldeath inhibitors can also be used to reduce or prevent cell death in thenervous system due to traumatic injury (such as head trauma), viralinfection or radiation-induced nerve cell death (for example, as aside-effect of cancer radiotherapy), as well as acute bacterialmeningitis (Braun et al., Nat Med 5:298-302 (1999)). The cell deathinhibitors can also be used to reduce or prevent cell death in a rangeof neurodegenerative disorders, including but not limited to Alzheimer'sdisease (Mattson et al., Brain Res. 807:167-176 (1998)), Huntington'sDisease, Parkinson's Disease, multiple sclerosis, amyotrophic lateralsclerosis, and spinobulbar atrophy. The in vivo neuroprotectiveproperties of cell death inhibitors of the invention can be tested in arat transient focal brain ischemia model (Xue et al., Stroke 21:166(1990)). The cell death inhibitors may also be used to treat orameliorate cell death in acute bacterial meningitis (Braun et al., NatMed 5:298-302 (1999)).

The cell death inhibitors of the invention can be used to reduce orprevent cell death in any condition which potentially results in thedeath of cardiac muscle (Black et al., J. Mol. Cel. Card. 30:733-742(1998) and Maulik et al., Free Radic. Biol. Med. 24:869-875 (1998)).This includes myocardial infarction due to myocardial ischemia andreperfusion, congestive heart failure and cardiomyopathy. One particularapplication is to reduce or prevent myocardial cell death as occurs incertain viral infections of the heart.

The in vivo activity of the cell death inhibitors of the invention canbe tested using the “mouse liver apoptosis” model described by Rodriguezet al. (J. Exp. Med. 184:2067-2072 (1996)). In this model, mice aretreated intravenously (IV) with an antiFas antibody which inducesmassive apoptosis in the liver and other organs, leading to generalizedorgan failure and death. This model is useful for indirectly testing thesystemic bioavailability of the cell death inhibitors of the invention,as well as their in vivo anti-apoptotic properties. The cell deathinhibitors of the invention therefore can be used to reduce or preventapoptosis of liver cells (Jones et al., Hepatology 27:1632-42 (1998))such as in sepsis (Jaeschke et al., J. Immunol. 160:3480-3486 (1998))and hereditary tyrosinemia type 1 (HT1) (Kubo et al., Prov. Natl. Acad.Sci. USA 95:9552-9557 (1998)). The cell death inhibitors of theinvention also can be used to treat hepatitis (Suzuki, Proc. Soc. Exp.Biol. Med. 217:450-454 (1998)).

The cell death inhibitors of the invention can be used to reduce orprevent cell death of retinal neurons (Kermer et al., J. Neurosci.18:4656-4662 (1998) and Miller et al., Am. J. Vet. Res. 59:149-152(1998)) as can occur in disorders which increase intraocular pressure(such as glaucoma) or retinal disorders associated with the agingprocess (such as age-related macular degeneration). The inhibitors canalso be used to treat hereditary degenerative disorders of the retina,such as retinitis pigmentosa.

The cell death inhibitors of the invention can also be used to reduce orprevent cell death in the kidney. This includes renal amyloidosis(Hiraoka et al., Nippon Jinzo Gakkai Shi. 40:276-83 (1998)), acute renalfailure (Lieberthal et al., Semin Nephrol. 18:505-518 (1998)), murinetubular epithelial cell death induced by cyclosporine A (Ortiz et al.,Kidney International Supp. 68:S25-S29 (1998)) and HIV-inducednephropathy (Conaldi et al., J. Clin. Invest. 102:2041-2049 (1998)).

The cell death inhibitors of the invention can also be used to reduce orprevent cell death of buccal mucosa due to chronic alcohol ingestion(Slomiany et al., Biochem. Mol. Biol. Int. 45:1199-1209 (1998)).

The cell death inhibitors of the invention can also be used to reduce orprevent cell death in plants (Richberg et al., Curr. Opin. Plant Biol.1:480-485 (1998)), such as plant cell death due to pathogens (Pozo etal., Curr. Biol. 8:1129-1132 (1998) and Greenberg et al., Cell77:551-563 (1994)).

The cell death inhibitors of the invention can also be used to reduce orprevent cell death due to radiation and ultraviolet-irradiation (Sheikhet al., Oncogene 17:2555-2563 (1998)).

The cell death inhibitors of the invention can also be used to reduce orprevent apoptotic death of bone marrow cells in myelodysplasticsyndromes (MDS) (Mundle et al., Am. J. Hematol. 60:36-47 (1999)).

The cell death inhibitors of the invention can also be used to reduce orprevent premature death of cells of the immune system, and areparticularly useful in treating immune deficiency disorders, such asacquired immune deficiency syndrome (AIDS), severe combined immunedeficiency syndrome (SCIDS) and related diseases. The cell deathinhibitors can also be used to treat radiation-induced immunesuppression.

Transplantation of human organs and tissues is a common treatment fororgan failure. However, during the transplantation process, the donororgan or tissue is at risk for cell death since it is deprived of itsnormal blood supply prior to being implanted in the host. This ischemicstate can be treated with cell death inhibitors by infusion into thedonor organ or tissue, or by direct addition of the cell deathinhibitors to the organ/tissue storage medium. For example, it wasreported that treatment of the embryonic nigral cell suspension withAc-YVAD-cmk (SEQ ID NO:2), a caspase-1 inhibitor, mitigated DNAfragmentation and reduced apoptosis in transplants. It also increasedsurvival of dopaminergic neurons grafted to hemiparkinsonian rats, andthereby substantially improved functional recovery (Schierle et al.,Nat. Med. 5:97-100 (1999)). Cell death inhibitors may also be used toreduce or prevent cell death in the donor organ/tissue after it has beentransplanted to protect it from the effects of reperfusion injury and/oreffects of host immune cells which kill their targets by triggeringapoptosis. The cytoprotective effects of cell death inhibitors can alsobe used to prevent the death of human or animal sperm and eggs used inin vitro fertilization procedures. These inhibitors can be used duringthe harvesting process and can also be included in the storage medium.

Mammalian cell lines, insect cells and yeast cells are commonly used toproduce large amounts of recombinant proteins (such as antibodies,enzymes or hormones) for industrial or medicinal use. The lifespan ofsome of these cell lines is limited due to growth conditions, the natureof the recombinant molecule being expressed (some are toxic) and otherunknown factors. The lifespans of industrial cell lines can be extendedby including these cell death inhibitors in the growth medium in aconcentration range of 1-100 μM.

The factors governing hair growth and loss are largely unknown. There issome evidence, however, that hair follicle regression (referred to ascatagen) may be due at least partially to apoptosis. Therefore, it iscontemplated that the cell death inhibitors of the present invention canbe used to treat hair loss that occurs due to various conditions,including but not limited to male-pattern baldness, radiation-induced orchemotherapy-induced hair loss, and hair loss due to emotional stress.There is also evidence that apoptosis may play a role in the loss ofhair color. Therefore, it is contemplated that the cell death inhibitorsof the present invention can also be used in treating or preventingcases of premature graying of the hair.

The death of skin epithelial cells can occur after exposure to highlevels of radiation, heat or chemicals. It is contemplated that the celldeath inhibitors of the present invention can be used to treat, reduceor prevent this type of skin damage. In one particular application, thecell death inhibitors can be applied as part of a topical formulation,e.g., an ointment, to treat acute over-exposure to the sun and toprevent blistering and peeling of the skin.

Goldberg et al. (Nature Genetics 13: 442-449 (1996)) reported recentlythat huntingtin, a protein product of Huntington's disease (HD) gene,can be cleaved by CPP32 but not ICE. The mutation underlying HD is anexpansion of a CAG trinucleotide at the 5′ end of the HD gene. Thetrinucleotide expansion exceeding 36 repeats is associated with theclinical presentation of HD. The CAG expansion promotes cleavage ofhuntingtin by CPP32, thus links the role of CPP32 in the apoptotic celldeath in HD. Compounds of the present invention with CPP32 inhibitingactivity will be useful in blocking CPP32 induced apoptotic cell death,thus in preventing and treating HD and other disorders characterized byexpansion of trinucleotide repeats such as myotonic dystrophy, fragile Xmental retardation, spinobulbar muscular atrophy, spinocerebellar atoxiatype I and Dentato-Rubro pallidoluysian atrophy.

The invention relates to a method of treating, ameliorating orpreventing oral mucositis, gastrointestinal mucositis, bladdermucositis, proctitis, bone marrow cell death, skin cell death and hairloss resulting from chemotherapy or radiation therapy of cancer in ananimal, comprising administering to the animal in need thereof aneffective amount of a cell death inhibitor of the present invention.

When animals are treated with chemotherapeutic agents and/or radiationto kill cancer cells, an unwanted side effect is the apoptotic death ofrapidly dividing non-cancer cells. Such non-cancer cells include cellsof the gastrointestinal tract, skin, hair, and bone marrow cells.According to the present invention, caspase inhibitors are administeredto such non-cancer cells to prevent apoptosis of such cells. In apreferred embodiment, the caspase inhibitors are administered locally,e.g., to the gastrointestinal tract, mouth, skin or scalp to preventapoptosis of the gastrointestinal, mouth, skin or hair cells butallowing for the death of the cancer cells. Thus, in one example, it ispossible to treat brain cancer with chemotherapy or radiation therapyand protect the outer skin, hair cells, gastrointestinal tract and bonemarrow by local administration of a caspase inhibitor. In the case oforal mucositis, the caspase inhibitor can be applied, for example, inthe form of a mouth wash or mouth rinse, in a gel, or in the form of anoral slow release lozenge to prevent activation of caspases andapoptotic cell death induced by the chemotherapeutic agent or byradiation. In the case of gastrointestinal mucositis, the caspaseinhibitor can be applied in a form such that it is not absorbedsystemically or in a form that coats the surface of the gastrointestinaltract, or a suppository formulation for the treatment ofgastrointestinal mucositis. In the case of proctitis, the capsaseinhibitor may be applied as part of an enema or suppository. In the caseof bladder mucositis, the caspase inhibitor may be applied though abladder catheter. For prevention of radiation or chemotherapy-inducedhair loss, the caspase inhibitor can be applied, for example, to thescalp in the form of a hair rinse, hair gel, shampoo or hairconditioner. Importantly, the caspase inhibitor can be applied prior tothe administration of the chemotherapeutic agent or radiation, thuspreventing the onset of the damaging effects of the chemotherapeuticagent or radiation to the normal cells.

Compositions within the scope of this invention include all compositionswherein the compounds of the present invention are contained in anamount which is effective to achieve its intended purpose. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typically, thecompounds may be administered to mammals, e.g., humans, orally at a doseof 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceuticallyacceptable salt thereof, per day of the body weight of the mammal beingtreated for apoptosis-mediated disorders, e.g., neuronal cell death,heart disease, retinal disorders, polycystic kidney disease, immunesystem disorders and sepsis. Preferably, about 0.01 to about 10 mg/kg isorally administered to treat or prevent such disorders. Forintramuscular injection, the dose is generally about one-half of theoral dose. For example, for treatment or prevention of neuronal celldeath, a suitable intramuscular dose would be about 0.0025 to about 25mg/kg, and most preferably, from about 0.01 to about 5 mg/kg.

The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.1 to about 10, conveniently about 0.25 to 50 mgof the compound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier. In a preferred embodiment,the compound is present at a concentration of about 0.07-1.0 mg/ml, morepreferably, about 0.1-0.5 mg/ml, most preferably, about 0.4 mg/ml.

In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally or topically and which canbe used for the preferred type of administration, such as tablets,dragees, slow release lozenges and capsules, mouth rinses and mouthwashes, gels, liquid suspensions, hair rinses, hair gels, shampoos andalso preparations which can be administered rectally, such assuppositories, as well as suitable solutions for administration byinjection, topically or orally, contain from about 0.01 to 99 percent,preferably from about 0.25 to 75 percent of active compound(s), togetherwith the excipient.

Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Acid addition salts are formed by mixing a solutionof the particular cell death inhibitors of the present invention with asolution of a pharmaceutically acceptable non-toxic acid such ashydrochloric acid, fumaric acid, maleic acid, succinic acid, aceticacid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalicacid, and the like. Basic salts are formed by mixing a solution of theparticular cell death inhibitors of the present invention with asolution of a pharmaceutically acceptable non-toxic base such as sodiumhydroxide, potassium hydroxide, choline hydroxide, sodium carbonate Trisand the like.

The pharmaceutical compositions of the invention may be administered toany animal which may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are mammals, e.g., humans,although the invention is not intended to be so limited.

The caspase inhibitors and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. In general, the caspase inhibitorsare administered locally to the tissues that are to be protected fromapoptosis and separately from the chemotherapeutic agent. For example,cisplatin may be administered by i.v. injection to treat a cancer suchas brain, lung, breast, liver, kidney, pancreatic, ovarian, prostaticcancer, and the caspase inhibitor administered locally to treat,ameliorate, or prevent apototic cell death in the mouth orgastrointestinal tract, such as a mouth wash for the treatment of oralmucositis; and IV injectable aqueous solution for the treatment of bonemarrow cell death; and an oral formulation suitable for coating thegastrointestinal surfaces or an emema or suppository formulation for thetreatment of gastrointestinal mucositis including proctitis. The caspaseinhibitors may also be applied through a bladder catheter for thetreatment, amelioration or prevention of bladder mucositis.Alternatively or concurrently, the caspase inhibitors may be appliedtopically to the skin and/or scalp to treat, ameliorate or preventapoptotic cell death of hair and skin cells. In a further embodiment,the chemotherapeutic agent or radiation may be applied locally to treata localized cancer such as brain, lung, breast, liver, kidney,pancreatic, ovarian, prostatic cancer, and the caspase inhibitoradministered systemically, e.g., by i.v. injection, to treat, ameliorateor prevent apoptotic cell death of the gastrointestinal tract cells,mouth epithelial cells, bone marrow cells, skin cells and hair cells. Inthe case of oral mucositis in brain cancer treatment, for example, acaspase inhibitor that does not penetrate the blood-brain barrier can beapplied, for example, systemically by i.v. injection followed byirradiation of the brain tumor. This would protect the oral mucosa fromthe harmful effects of radiation but the caspase inhibitor would notprotect the brain tumor from the therapeutic effects of radiation.Importantly, the caspase inhibitor can be applied prior toadministration of the radiation, thus preventing the onset of thedamaging effects of the radiation to the normal mucosa cells.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, enemas or suppositories, which consist of a combination ofone or more of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400 (thecompounds are soluble in PEG-400). Aqueous injection suspensions maycontain substances which increase the viscosity of the suspensioninclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.

In accordance with one aspect of the present invention, compounds of theinvention are employed in topical and parenteral formulations and areused for the treatment of skin damage, such as that caused by exposureto high levels of radiation, including ultraviolet radiation, heat orchemicals.

One or more additional substances which have therapeutic effects on theskin may also be incorporated in the compositions. Thus, the compositionmay also contain one or more compounds capable of increasing cyclic-AMPlevels in the skin. Suitable compounds include adenosine or a nucleicacid hydrolysate in an amount of about 0.1-1% and papaverine, in anamount of about 0.5-5%, both by weight based on the weight of thecomposition. Also suitable are β-adrenergic agonists such asisoproterenol, in an amount of about 0.1-2% or cyclic-AMP, in an amountof about 0.1-1%, again both by weight based on the weight of thecomposition. Other suitable types of additional active ingredients whichmay be incorporated in the compositions of this invention include anycompounds known to have a beneficial effect on skin. Such compoundsinclude retinoids such as Vitamin A, in an amount of about 0.003-0.3% byweight and chromanols such as Vitamin E or a derivative thereof in anamount of about 0.1-10% by weight, both based on the weight of thecomposition. Additionally, anti-inflammatory agents and keratoplasticagents may be incorporated in the cosmetic composition. A typicalanti-inflammatory agent is a corticosteroid such as hydrocortisone orits acetate in an amount of about 0.25-5% by weight, or a corticosteroidsuch as dexamethasone in an amount of about 0.025-0.5% by weight, bothbased on the weight of the composition. A typical keratoplastic agent iscoal tar in an amount of about 0.1-20% or anthralin in an amount ofabout 0.05-2% by weight, both based on the weight of the composition.

The topical compositions of this invention are formulated preferably asoils, creams, lotions, ointments and the like by choice of appropriatecarriers. Suitable carriers include vegetable or mineral oils, whitepetrolatum (white soft paraffin), branched chain fats or oils, animalfats and high molecular weight alcohol (greater than C₁₂). The preferredcarriers are those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil such as almond oil, isadmixed. A typical example of such a cream is one which includes about40 parts water, about 20 parts beeswax, about 40 parts mineral oil andabout 1 part almond oil.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight.

Lotions may be conveniently prepared by dissolving the activeingredient, in a suitable high molecular weight alcohol such aspropylene glycol or polyethylene glycol.

In addition, these compositions may include other medicinal agents,growth factors, wound sealants, carriers, etc., that are known orapparent to those skilled in the art. The compositions of the inventionare administered to a warm-blooded animal, such as human, alreadysuffering from a skin damage, such as a burn, in an amount sufficient toallow the healing process to proceed more quickly than if the host werenot treated. Amounts effective for this use will depend on the severityof the skin damage and the general state of health of the patient beingtreated. Maintenance dosages over a prolonged period of time may beadjusted as necessary. For veterinary uses, higher levels may beadministered as necessary.

In the case of an animal suffering from decreased hair growth, thecompositions of the invention are administered in an amount sufficientto increase the rate of hair growth. Amounts effective for this use willdepend on the extent of decreased hair growth, and the general state ofhealth of the patient being treated. Maintenance dosages over aprolonged period of time may be adjusted as necessary. For veterinaryuses, higher levels may be administered as necessary.

When the compounds are to be administered to plants, they may be appliedto the leaves and/or stems and/or flowers of the plant, e.g., byspraying. The compounds may be spayed in particulate form or dissolvedor suspended in an appropriate carrier, e.g., in water or an oil wateremulsion. The compounds may also be combined with the soil of the plant.In this embodiment, the compounds are taken up by the roots of theplant.

In a preferred embodiment, the caspase inhibitor is formulated as partof a mouthwash for the treatment, amelioration or prevention of oralmucositis. Such mouthwashes are aqueous solutions of the caspaseinhibitor which may also contain alcohol, glycerin, synthetic sweetenersand surface-active, flavoring and coloring agents. They may also containanti-infective agents such as hexetidine and cetylpyridinium chloride.The mouthwashes may also contain topical anesthetics (e.g., benzocaine,cocaine, dyclonine hydrochloride, lidocaine, proparacaine hydrochlorideor teracaine hydrochloride), for example, for relieving pain ofradiation or chemotherapy-induced sores. The mouth washes may haveeither acidic or basic pH. See Remington's Pharmaceutical Sciences, A.R. Gennaro (ed.), Mack Publishing Company, pp. 1045, 1046, 1526 and 1965(1990).

In another preferred embodiment, the caspase inhibitor is formulated asan oral formulation which is capable of coating the gastrointestinalsurfaces for the treatment, amelioration or prevention ofgastrointestinal mucositis. Examples of gastrointestinal mucositisinclude esophageal mucositis, gastric mucositis, and intestinalmucositis. Such formulations may comprise gastric antacids such asaluminum carbonate, aluminum hydroxide gel, bismuth subnitrate, bismuthsubsalicylate, calcium carbonate, dihydroxyaluminum sodium carbonate,magaldrate, magnesium carbonate, magnesium hydroxide, magnesium oxide,sodium bicarbonate, milk of bismuth, dihydroxyaluminum aminoacetate,magnesium phosphate, magnesium trisilicate and mixtures thereof. Otheradditives include without limitation H₂-receptor antagonists,digestants, anti-emetics, adsorbants, and miscellaneous agents. SeeRemington's Pharmaceutical Sciences, A. R. Gennaro (ed.), MackPublishing Company, pp. 774-778 (1990).

Chemotherapy agents such as cisplatin and radiation therapy often induceearly and late onset emesis in the patient. Thus, in one embodiment anantiemetic is coadminstered together with the caspase inhibitor to avoidemesis and retain contact of the caspase inhibitor with thegastrointestinal tract. Examples of such antiemetics include withoutlimitation compounds that block the dopaminergic emetic receptors suchas metoclopramide and trimethobenzamide, and cannabinoids.Metoclopramide may be administered orally prior to and/or duringchemotherapy/radiation therapy/caspase inhibitor therapy to prevent theearly emesis response and then later by intranasal administrationaccording to U.S. Pat. Nos. 5,760,086 and 4,536,386 to prevent delayedonset emesis. During the period after chemotherapy/radiation therapy,both the caspase inhibitor and the antiemetic may be coadministered totreat, ameliorate or prevent gastrointestinal mucositis.

In a further embodiment, the caspase inhibitor may be formulated as anIV injectable solution for the treatment, amelioration or prevention ofbone marrow cell death.

The compositions of the invention may be administered to a warm-bloodedanimal, such as human, already suffering from chemotherapy or radiationtherapy-induced non-cancer cell death, or, more preferably, before orduring therapy with chemotherapy or radiation.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLE 1 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk Step A. 2-Chlorobenzylchloroformate

To a solution of 2-chlorobenzyl alcohol (1.0 g, 7.0 mmol) in diethylether (15 ml) at 0° C. was added N,N-diisopropylethyl amine (2.4 ml,14.0 mmol), and phosgene solution in toluene (7.5 ml, 14.0 mmol). Themixture was allowed to warm up to room temperature in 2 h whilestirring, then it was filtered. The diethyl ether was removed by rotaryevaporator, and the solution of 2-chlorobenzyl chloroformate in toluenewas carried on for the next step reaction.

Step B. 2-Chlorobenzyloxycarbonyl-Val

To a solution of L-valine (0.5 g, 4.3 mmol) in 2 N NaOH aqueous solution(10 ml) was added 2-chlorobenzyl chloroformate (14.0 mmol) at roomtemperature. The resulting solution was stirred at room temperature for12 h, and then was diluted with 20 ml of ethyl acetate, washed with 2NNaOH, 2N HCl and brine, dried over Na₂SO₄ and concentrated in vacuo. Thetitle compound was obtained as white solid (0.94 g, 3.29 mmol, 77%). ¹HNMR (DMSO-d₆): 12.24 (bs, 1 H), 7.61 (d, J=8.4, 1 H), 7.50 (m, 2 H),7.37 (m, 2 H), 5.12 (s, 2 H), 3.87 (dd, J=8.7, 6.0, 1 H), 2.06 (m, 1 H),0.89 (m, 6 H).

Step C. t-Butyl5-fluoro-3-(2-chlorobenzyloxycarbonyl-valinamido)-4-hydroxypentanoate

To a solution of 2-chlorobenzyl-oxycarbonyl-Val (216 mg, 0.76 mmol) inTHF (10 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDCI) (159 mg, 0.83 mmol), 1-hydroxybenzotriazole hydrate(HOBT) (116 mg, 0.77 mmol) and 4-(dimethylamino)pyridine (DMAP) (46 mg,0.38 mmol). The resulting mixture was stirred at room temperature for 5min, to which was then added a solution of t-butyl3-amino-5-fluoro-4-hydroxypentanoate (157 mg, 0.75 mmol) in THF (5 ml).The resulting mixture was stirred at room temperature for 12 h, anddiluted with ethyl acetate (20 ml), washed with 1N HCl, saturatedNaHCO₃, brine and dried over Na₂SO₄. Evaporation of solvent, followed byflash chromatography (EtOAc/Hexane 2/3) gave the title compound as acolorless oil (80 mg, 0.17 mmol, 22%). ¹H NMR (CDCl₃): 7.38 (m, 2 H),7.27 (m, 2 H), 7.06-6.84 (m, 1 H), 5.56 (m, 1 H), 5.22 (s, 2 H),4.51-4.21 (m, 3 H), 4.01 (m, 3 H), 2.63 (m, 2 H), 2.12 (m, 1 H), 1.43(m, 9H), 0.96 (m, 6H).

Step D. 2-Chlorobenzyloxycarbonyl-Val-Asp(OBu-t)-fmk

To a suspension of Dess-Martin periodinane (0.35 g, 0.835 mmol) indichloromethane (15 ml) was added a solution of t-butyl5-fluoro-3-(2-chlorobenzyloxycarbonyl-valinamido)-4-hydroxypentanoate(80 mg, 0.17 mmol) in dichloromethane (5 ml). The mixture was refluxedfor 12 h, cooled to room temperature, then it was diluted with 25 ml ofethyl acetate, washed with saturated Na₂SO₃ aqueous solution, brine, andthen dried over Na₂SO₄. Evaporation of the solvent, followed by flashchromatography (EtOAc/Hexane 1/2) gave the title compound as a palewhite solid (56 mg, 0.12 mmol, 72%). ¹H NMR (CDCl₃): 7.39 (m, 2 H), 7.28(m, 2 H), 5.43 (m, 1 H), 5.25-4.84 (m, 5 H), 4.05 (m, 1 H), 2.96 (m, 1H), 2.77 (m, 1 H), 2.14 (m, 1 H), 1.42 (s, 9H), 0.96 (m, 6H).

Step E. 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk

To a solution of 2-chlorobenzyloxycarbonyl-Val-Asp(OBu-t)-fmk (56 mg,0.12 mmol) in 3 ml of CH₂Cl₂ at room temperature was added 1 ml of TFA.The resulting solution was allowed to stir for 3 h, and then dilutedwith 20 ml of ethyl acetate, washed with saturated Na₂HPO₄, brine, anddried over Na₂SO₄. The solvent was evaporated in vacuo to give the titlecompound as a white solid (39 mg, 0.09 mmol, 77%). ¹H NMR (CDCl₃): 8.61(m, 1 H), 8.15 (m, 1 H), 7.49 (m, 2 H), 7.37 (m, 2 H), 5.21 (m, 1 H),5.11 (s, 2 H), 4.59 (m, 2 H), 3.84 (m, 1 H), 2.65 (m, 2 H), 1.98 (m,1H), 0.86 (m, 6 H).

EXAMPLE 2 3-Chlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3-chlorobenzyl alcohol. ¹H NMR (CDCl₃): 7.55 (bs, 1 H), 5.48 (m, 1H), 4.90 (m, 4 H), 4.02 (m, 2 H), 3.85 (m, 1 H), 3.08 (m, 1 H), 2.76 (m,1 H), 1.93 (m, 1 H), 1.71 (m, 7 H), 1.23 (m, 4 H ), 0.95 (m, 6 H).

EXAMPLE 3 Phenethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from phenethyl alcohol. ¹H NMR (CDCl₃): 7.87 (m, 2 H), 7.23 (m, 5 H),5.74 (d, J=8.4, 1 H), 4.87 (m, 2 H), 4.26 (m, 3 H), 3.99 (m, 1 H), 2.89(m, 4 H), 2.09 (m, 1 H), 0.91 (m, 6 H).

EXAMPLE 4 4-Chlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 4-chlorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.51 (m, 1 H), 7.42 (m, 5H), 5.21 (m, 1 H), 5.03 (s, 2 H), 4.60 (m, 1 H), 3.83 (m, 1 H), 2.65 (m,2 H), 1.93 (m, 1 H), 0.85 (m, 6 H).

EXAMPLE 5 Cyclohexylmethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from cyclohexylmethanol. ¹H NMR (CDCl₃): 7.55 (bs, 1 H), 5.48 (m, 1 H),4.90 (m, 4 H), 4.02 (m, 2 H), 3.85 (m, 1 H), 3.08 (m, 1 H), 2.76 (m, 1H), 1.93 (m, 1 H), 1.71 (m, 7 H), 1.23 (m, 4 H), 0.95 (m, 6 H).

EXAMPLE 6 Ethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in four steps as described in Example 1from L-valine and ethyl chloroformate. ¹H NMR (DMSO-d₆): 8.43 (s, 1H),7.19 (m, 1H), 5.14(bs, 2H), 4.66-4.53(m, 1H), 4.01(q, J=6.9, 2H),3.83-3.76 (m, 1H), 2.73-2.67 (m, 2H), 1.96-1.88 (m, 1H), 1.16 (t, J=6.9,3H), 0.86-0.82 (m, 6H).

EXAMPLE 7 Benzylcarbonyl-Val-Asp-fmk

The title compound was prepared in four steps as described in Example 1from phenylacetyl chloride. ¹H NMR (DMSO-d₆): 8.55 (m, 1H), 8.21 (m,1H), 7.21 (m, 5H), 5.14 (m, 2H), 4.62 (m, 2H), 4.14 (m, 1H), 2.66 (m,2H), 1.93 (m, 1H), 0.83 (m, 6H).

EXAMPLE 8 4-Nitrobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in four steps as described in Example 1from 4-nitrobenzyl chloroformate. ¹H NMR (DMSO-d₆): 12.5 (s, 1H),8.66-8.54 (m, 1H), 8.23 (m, 2H), 7.64 (m, 2H), 5.19 (m, 4H), 4.66-4.54(m, 2H), 3.82 (m, 1H), 2.76 (m, 2H), 1.95 (m, 1H), 0.87 (m, 6H).

EXAMPLE 9 2,5-Dimethylbenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2,5-dimethylbenzyl alcohol. ¹H NMR (DMSO-d₆): 8.50 (m, 1H), 7.42(m, 1H), 7.06 (m, 2H), 4.99 (s, 2H), 4.64-4.56 (m, 1H), 3.83 (m, 1H),2.97 (m, 1H), 2.67 (m, 2H), 1.89 (m, 1H), 0.86 (m, 6H).

EXAMPLE 10 3,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3,4-dichlorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.50 (m, 1H),7.65-7.35 (m, 4H), 5.03 (m, 3H), 4.59 (m, 1H), 3.84 (m, 1H), 2.67 (m,2H), 1.94 (m, 1H), 0.86 (m, 6H).

EXAMPLE 11 3,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3,5-dichlorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.54 (m, 1H),7.56-7.34 (m, 3H), 5.05 (m, 3H), 4.63-4.55 (m, 1H), 3.86 (m, 1H), 2.73(m, 2H), 1.95 (m, 1H), 0.86 (m, 6H).

EXAMPLE 12 2,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2,5-dichlorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.54 (m, 1H), 7.51(m, 3H), 5.26-5.08 (m, 3H), 4.65-4.55 (m, 1H), 3.87 (m, 1H), 2.73-2.60(m, 2H), 1.98 (m, 1H), 0.86 (m, 6H).

EXAMPLE 13 2,6-Dichlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2,6-dichlorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.50 (m, 1H),7.55-7.41 (m, 3H), 5.25 (m, 3H), 4.64-4.51 (m, 1H), 3.78 (m, 1H), 2.71(m, 2H), 1.92 (m, 1H), 0.84 (m, 6H).

EXAMPLE 14 2,4-Dimethylbenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2,4-dimethylbenzyl alcohol. ¹H NMR (DMSO-d₆): 8.53 (m, 1H), 7.39(m, 1H), 7.28-7.18 (m, 3H), 5.26 (m, 1H), 4.99 (m, 2H), 4.66-4.53 (m,1H), 3.80 (m, 1H), 2.78-2.72 (m, 1H), 2.59 (m, 3H), 1.92 (m, 1H), 1.16(m, 3H), 0.85 (m, 6H).

EXAMPLE 15 4-Ethylbenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 4-ethylbenzyl alcohol. ¹H NMR (DMSO-d₆): 8.50 (m, 1H), 7.23 (m,4H), 4.98 (m, 3H), 4.63 (m, 1H), 3.84 (m, 1H), 2.68 (m, 2H), 1.91 (m,1H), 0.85 (m, 6H).

EXAMPLE 16 4-Chlorobenzloxycarbonyl-Ile-Asp-fmk

The title compound was prepared in five steps as described in example 1from 4-chlorobenzyl alcohol. ¹H NMR (CDCl₃): 8.61 (m, 1H), 8.52 (m, 1H),7.48 (m, 1H), 7.39 (m, 4H), 5.19 (m, 2H), 5.02 (s, 2H), 4.54 (m, 1H),3.87 (m, 1H), 2.73 (m, 2H), 1.68 (m, 1H), 1.39 (m, 1H), 1.17 (m, 1H),0.81 (m, 6H).

EXAMPLE 17 4-Bromobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in example 1from 4-bromobenzyl alcohol. ¹H NMR (DMSO-d6): 7.56 (d, J=8.1 Hz, 1H),7.42 (m, 1H), 7.32 (d, J=8.1 Hz, 1H), 5.21 (m, 1H), 5.00 (s, 2H), 4.62(m, 2H), 3.83 (m, 1H), 3.60 (m, 1H), 2.71 (m, 2H), 1.76 (m, 1H), 0.84(m, 6H).

EXAMPLE 18 4-Fluorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 4-fluorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.63 (m, 1H), 7.92 (m,1H), 7.55 (m, 4H), 5.03 (m, 3H), 4.61 (m, 2H), 3.83 (m, 1H), 2.73 (m,2H), 1.95 (m, 1H), 0.95 (m, 6H).

EXAMPLE 19 2,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2,4-dichlorobenzyl alcohol. ¹H NMR (CDCl₃): 8.51 (m, 1H), 7.52 (m,3H), 5.08 (m, 3H), 4.59 (m, 2H), 3.85 (m, 1H), 2.69 (m, 2H), 1.94 (m,1H), 0.95 (m, 6H).

EXAMPLE 20 Cyclopentylmethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from cyclopentylmethanol. ¹H NMR (DMSO-d₆): 7.22 (bs, 1H), 5.21 (m, 1H),5.09 (m, 1H), 4.61 (m, 2H), 3.84 (m, 3H), 2.73 (m, 2H), 2.10 (m, 1H),1.92 (m, 1H), 1.49 (m, 6H), 1.23 (m, 2H), 0.84 (m, 6H).

EXAMPLE 21 4-Trifluoromethylbenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in four steps as described in Example 1from 4-trifluoromethylbenzyl alcohol. ¹H NMR (DMSO-d₆): 8.63 (m, 1H),8.18 (m, 1H), 7.74 (d, J=7.2 Hz, 2H), 7.57(d, J=8.1, 2H), 5.14 (m, 4H),4.64 (m, 1H), 3.81 (m, 1H), 2.72 (m, 2H), 1.94 (m, 1H), 0.86 (m, 6H).

EXAMPLE 22 3-Phenylpropionyl-Val-Asp-fmk

The title compound was prepared in four steps as described in Example 2from 3-phenylpropionyl chloride. ¹H NMR (DMSO-d₆): 12.29 (bs, 1H), 8.39(m, 1H), 7.82 (m, 1H), 7.21(m, 5H), 5.25 (m, 2H), 4.39 (m, 1H), 4.03 (m,1H), 2.80 (m, 3H), 2.54 (m, 3H), 1.89 (m, 1H), 0.80 (m, 6H).

EXAMPLE 23 Benzylaminocarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from benzyl amine. ¹H NMR (DMSO-d₆): 8.55 (m, 1H), 7.25 (m, 5H), 6.57(m, 1H), 6.20 (m, 1H), 5.16 (m, 1H), 4.22 (m, 5H), 2.61 (m, 2H), 1.89(m, 1H), 0.87 (m, 6H).

EXAMPLE 24 3-Phenylpropyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3-phenyl-1-propanol. ¹H NMR (DMSO-d₆): 8.61 (m, 1H), 8.18 (m, 1H),7.21 (m, 5H), 5.17 (m, 1H), 4.53 (m, 1H), 3.83 (m, 4H), 2.71 (m, 4H),1.85 (m, 3H), 0.85 (m, 6H).

EXAMPLE 25 2,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2,4-difluorobenzyl alcohol. ¹H NMR (CDCl₃): 7.33 (m, 1H), 6.83 (m,2H), 5.60 (m, 1H), 5.11 (m, 3H), 4.88 (m, 2H), 4.03 (m, 1H), 3.05 (m,1H), 2.79 (m, 1H), 2.05 (m, 1H), 0.94 (m, 6H).

EXAMPLE 26 3,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3,4-difluorobenzyl alcohol. ¹H NMR (CDCl3): 8.63 (m, 1H), 8.18 (m,1H), 7.52 (m, 1H), 7.27 (m, 2H), 5.07 (m, 3H), 4.51 (m, 2H), 3.99 (m,1H), 2.70 (m, 2H), 1.95 (m, 1H), 0.85 (m, 6H).

EXAMPLE 27 4-Morpholinecarbonyl-Val-Asp-fmk

The title compound was prepared in four steps as described in Example 1from 4-morpholinecarbonyl chloride. ¹H NMR (CDCl₃): 10.61 (bs, 1H), 7.58(m, 1H), 5.19 (d, J=8.1 Hz, 1H), 4.95 (m, 3H), 4.42 (m, 1H), 3.71 (m,4H), 3.42 (m, 4H), 2.94 (m, 2H), 2.19 (m, 1H), 0.98 (m, 6H).

EXAMPLE 28 4-Pyridylmethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 4-pyridylcarbinol. ¹H NMR (CDCl3): 8.56 (m, 2H), 8.15 (m, 2H), 7.23(m, 1H), 6.47 (m, 1H), 5.10 (s, 2H), 4.35 (m, 3H), 4.08 (m, 1H), 2.61(m, 2H), 1.46 (m, 1H), 0.94 (m, 6H).

EXAMPLE 29 2-Pyridylmethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2-pyridylcarbinol. ¹H NMR (CDCl₃): 8.59 (m, 1H), 7.28 (m, 3H), 7.03(m, 1H), 5.58 (m, 1H), 5.11 (m, 5 H), 4.06 (m, 1H), 2.83 (m, 2H), 2.17(m, 1H), 0.93 (m, 6H).

EXAMPLE 30 2,6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone

Step A. Z-Asp(OBu-t)-DCB-methylketone

To a solution of Z-Asp(OBu-t)-bromomethylketone (500 mg, 1.24 mmol) inDMF (10 ml) was added potassian fluoride (320 mg, 5.50 mmol), and2,6-dichlorobenzoic acid (348 mg, 1.82 mmol). The mixture was stirred atroom temperature for 12 h, and then was diluted with 25 ml of ethylacetate, washed with aqueous NH₄Cl and brine, dried over Na₂SO₄ andconcentrated in vacuo. The title compound was obtained as white solid(0.78 g, 2.62 mmol, 69%). ¹H NMR (CDCl₃): 7.34 (m, 8 H), 5.96 (d, J=8.7,1H), 5.21 (d, J=6.6, 2H), 5.16 (s, 2H), 4.70 (m, 1H), 2.88 (m, 2H), 1.27(s, 9 H).

Step B. Asp(OBu-t)-DCB-methylketone-N-hydrochloride

To a solution of Z-Asp(OBu-t)-DCB-methylketone (572 mg, 1.14 mmol) inethanol (15 ml) was added Pd/C (50 mg) and 6N HCl (0.2 ml). The mixturewas stirred at room temperature under H₂ atmosphere (1 atm) for 12 h,then it was filtered and concentrated. The title compound was obtainedas pale while solid (416 mg, 1.04 mmol, 90%). ¹H NMR (CDCl₃): 7.27 (m,3H), 5.28 (m, 2H), 4.94 (m, 1H), 3.27 (m, 2H), 1.42 (s, 9 H).

Step C. 2,6-Dichlorobenzyloxycarbonyl-Val-Asp(OBu-t)-DCB-methylketone

To a solution of 2,6-dichlorobenzyloxycarbonyl-Val (200 mg, 0.60 mmol)in THF (10 ml) was added Asp(OBu-t)-DCB-methylketone-N-hydrochloride(250 mg, 0.60 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDCI, 126 mg, 0.66 mmol), 1-hydroxybenzotriazole hydrate(HOBT, 92 mg, 0.60 mmol) and 4-(dimethylamino)pyridine (DMAP, 29 mg,0.28 mmol). The resulting mixture was stirred at room temperature for 12h, and diluted with ethyl acetate (20 ml), washed with 1N HCl, saturatedNaHCO₃, brine and dried over Na₂SO₄. Evaporation of solvent, followed byflash chromatography (EtOAc/Hexane 2/3) gave the title compound as acolorless oil (85 mg, 0.18 mmol, 21%). ¹H NMR (CDCl₃): 7.35 (m, 6H),7.27 (m, 1H), 5.41 (m, 3H), 5.15 (m, 2H), 4.41 (m, 1H), 2.96 (m, 2H),2.28 (m, 1H), 1.44 (m, 9 H), 0.94 (m, 6H).

Step D. 2,6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone

To a solution of2,6-dichlorobenzyloxycarbonyl-Val-Asp(OBu-t)-DCB-methylketone (85 mg,0.18 mmol) in CH₂Cl₂ (3 ml) at room temperature was added TFA (1 ml).The resulting solution was allowed to stir for 4 h, and then dilutedwith 20 ml of ethyl acetate, washed with saturated Na₂HPO₄, brine, anddried over Na₂SO₄. The solvent was evaporated in vacuo to give the titlecompound as a white solid (27 mg, 0.04 mmol, 25%). ¹H NMR (CDCl₃): 9.28(bs, 1H), 8.11 (m, 1H), 7.49 (m, 1H), 7.30 (m, 6H), 5.37(m, 4H), 4.32(m, 2H), 3.73 (m, 2H), 2.19 (m, 1H), 1.25 (m, 3H), 0.98 (m, 3H).

EXAMPLE 31 Isobutoxycarbonyl-Val-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from L-valine and isobutyl chloroformate. ¹H NMR (DMSO-d₆): δ8.47 (m,1H), 7.23 (m, 1H), 5.24-4.52 (m, 3H), 3.81-3.72 (m, 3H), 2.73-2.55 (m,2H), 1.94-1.79 (m, 2H), 0.89-0.83 (m, 12H).

EXAMPLE 32 Methoxycarbonyl-Val-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from L-valine and methyl chloroformate. ¹H NMR (DMSO-d₆): δ8.49 (s, 1H),7.28 (m, 1H), 5.16-4.54 (m, 3H), 3.81 (m, 1H), 3.53 (s, 3H), 2.72-2.56(m, 2H), 1.93 (m, 1H), 0.85 (m, 6H).

EXAMPLE 33 Isopropyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from L-valine and isopropyl chloroformate. ¹H NMR (DMSO-d₆): δ8.43 (s,1H), 7.11 (m, 1H), 5.18-4.54 (m, 4H), 3.76 (m, 1H), 2.69-2.67 (m, 2H),1.91 (m, 1H), 1.17 (d, J=6, 6H), 0.83 (m, 6H).

EXAMPLE 34 Propionyl-Val-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from L-valine and propionyl chloride. ¹H NMR (DMSO-d₆): δ8.63-7.82 (m,2H), 5.37-5.02 (m, 1H), 4.70-3.95 (m, 3H), 2.89-2.56 (m, 2H), 2.21-2.10(m, 2H), 1.91 (m, 1H), 0.98 (t, J=7.2, 3H), 0.86-0.82 (m, 6H).

EXAMPLE 35 Benzyl-glutaryl-Val-Asp-fmk Step A. Benzyl monoglutarate

A solution of glutaric anhydride (460 mg, 4.03 mmol) in benzyl alcohol(1 ml) was heated at 70° C. overnight. The mixture was purified bychromatography (3:1 hexane/EtOAc) to yield the product as an oil (0.4 g,1.8 mmol, 45%). ¹H NMR (CDCl₃): δ7.36 (br s, 5H), 5.12 (s, 2H),2.48-2.41 (m, 4H), 2.00-1.96 (m, 2H).

Step B. Benzyl-glutaryl-Val-OBu-t

A mixture of benzyl monoglutarate (0.4 g, 1.8 mmol), Val-OBu-t (382 mg,1.82 mmol), EDC (335 mg, 1.75 mmol), HOBT (265 mg, 1.73 mmol) and DMAP(372 mg, 3.04 mmol) in THF (15 ml) was stirred at room temperature for17 h. It was worked up and purified by chromatography to yield the titlecompound as a colorless oil (370 mg, 0.98 mmol, 54%). ¹H NMR (CDCl₃):δ7.39-7.30 (m, 5H), 5.96 (d, J=8.4, 1H), 5.12 (s, 2H), 4.45 (m, 1H),2.47-1.94 (m, 7H), 1.46 (s, 9H), 0.95-0.87 (m, 6H).

Step C. Benzyl-glutaryl-Val-OH

To a solution of benzyl-glutaryl-Val-OBu-t (370 mg, 0.98 mmol) inmethylenechloride (3 ml) was added TFA (1 ml). The mixture was stirredat room temperature for 8 h. It was worked up to yield the titlecompound as a colorless oil (200 mg, 0.62 mmol, 63%). ¹H NMR (CDCl₃):δ7.36 (br s, 5H), 6.43 (d, J=8.7, 1H), 5.13 (s, 2H), 4.55 (m, 1H),2.49-2.21 (m, 5H), 2.00 (m, 2H), 0.99-0.94 (m, 6H).

Step D. Benzyl-glutaryl-Val-Asp-fmk

The title compound was prepared from benzyl-glutaryl-Val-OH in threesteps by a similar procedure as described in Example 1 as a white solid.¹H NMR (CDCl₃): δ7.35 (br s, 5H), 6.72 (s, 1H), 5.56 (s, 1H), 5.11 (s,2H), 4.92-4.22 (m, 4H), 3.13-2.68 (m, 2H), 2.43-1.93 (m, 6H), 0.92 (d,J=5.1, 6H).

EXAMPLE 36 Glutaryl-Val-Asp-fmk

A solution of benzyl-glutaryl-Val-Asp(OBu-t)-fmk (78 mg, 0.15 mmol) in30% AcOH solution of HBr (2 ml) was stirred at room temperature for 4 h.The solvent was removed in vacuo. To the residue was added acetone (1ml), then EtOAc (10 ml) and hexane (20 ml). The solvents were thenremoved in vacuo to yield the title compound as a brown solid (36 mg,0.10 mmol, 67%). ¹H NMR (DMSO-d₆): δ8.60-7.87 (m, 2H), 5.36-4.42 (m,4H), 2.94-2.57 (m, 2H), 2.33-1.69 (m, 7H), 0.83 (s, 6H).

EXAMPLE 37 3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk Step A.3-(2-Phenyloxyphenyl)propionic acid

A mixture of 3-(2-hydroxyphenyl)propionic acid (0.57 g, 3.4 mmol),phenylbromide (0.4 ml, 3.37 mmol), and K₂CO₃ (2.5 g) in acetone (10 ml)was stirred at room temperature for 2 days. The mixture was diluted with1:1 hexane/EtOAc (100 ml), washed with water and brine, dried oversodium sulfate and concentrated in vacuo. The residue was purified bychromatography (5:1 then 4:1 hexane/EtOAc) to yield phenyl3-(2-phenyloxyphenyl)propionate (0.45 g, 1.30 mmol, 77%).

A mixture of phenyl 3-(2-phenyloxyphenyl)propionate (0.45 g, 1.30 mmol),2N NaOH (20 ml) and MeOH (10 ml) was stirred at room temperatureovernight, which was then neutralized to pH˜2 with concentrated HCl andextracted with EtOAc (100 ml). The organic phase was washed with brineand dried over sodium sulfate and concentrated in vacuo to yield3-(2-phenyloxyphenyl)propionic acid as an oil. This was used for nextstep without further purification.

Step B. 3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk

The title compound was prepared in a similar procedure as described inExamples 36 and 1 from crude 3-(2-phenyloxyphenyl)propionic acidsynthesized above. ¹H NMR (DMSO-d₆): δ8.62-8.50 (m, 1H), 7.47-6.85 (m,9H), 5.36-5.04 (m, 4H), 4.63-4.02 (m, 2H), 5.83 (br s, 6H), 1.85 (br s,1H), 0.80 (s, 6H).

EXAMPLE 38 3-(5-Bromo-2-hydroxyphenyl)propionyl-Val-Asp-fmk

A mixture of 3-(2-phenyloxyphenyl)propionyl-Val-Asp-fmk (30 mg, 30 mg,0.06 mmol) in 30% acetic acid solution of HBr (2 ml) was stirred at roomtemperature for 6 h. The solvent was removed in vacuo and 1:1EtOAc/hexane was added to the residue. Evaporation of the solventyielded the title compound as a brown solid (18 mg, 0.037 mmol, 62%). ¹HNMR (acetone-d₆): δ7.45-6.74 (m, 4H), 5.03-4.35 (m, 4H), 3.03-2.73 (m,6H), 0.97-0.87 (m, 6H).

EXAMPLE 39 3-Fluorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3-fluorobenzyl alcohol. ¹H NMR (DMSO-d₆): 8.64 (m, 1H), 7.95 (m,1H), 7.45 (m, 2H), 7.19 (m, 3H), 5.25 (m, 1H), 5.06 (s, 2H), 4.58 (m,2H), 3.85 (m, 1H), 2.73 (m, 2H), 1.99 (m, 1H), 0.86 (m, 6H).

EXAMPLE 40 2-Fluorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 2-fluorobenzyl alcohol. ¹H NMR (DMSO-d₆): 7.36 (m, 2H), 7.12 (m,3H), 5.68 (m, 1H), 5.17 (s, 2H), 4.34 (m, 3H), 3.97 (m, 2H), 2.62 (m,2H), 2.06 (m, 1H), 0.91 (m, 6H).

EXAMPLE 41 3-Methylbenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 1from 3-methylbenzyl alcohol. ¹H NMR (DMSO-d₆): 8.52 (m, 1H), 7.92 (m,1H), 7.43 (m, 1H), 7.17 (m, 4H), 5.25 (m, 1H), 4.99 (s, 2H), 4.56 (m,2H), 3.83 (m, 1H), 2.71 (m, 2H), 2.29 (s, 3H), 1.93 (m, 1H), 0.85 (m,6H).

EXAMPLE 42 2-Chloro-4-fluorobenzyloxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 2from 2-chloro-4-fluorobenzyl alcohol. ¹H NMR (CDCl3): 8.63 (m, 1H), 7.42(m, 4H), 5.15 (m, 3H), 4.61 (m, 2H), 4.02 (bs, 1H), 3.81 (m, 1H), 2.63(m, 2H), 1.90 (m, 1H), 0.83 (m, 6H).

EXAMPLE 43 2-Naphthalenemethoxycarbonyl-Val-Asp-fmk

The title compound was prepared in five steps as described in Example 2from 2-naphthalenemethanol. ¹H NMR (DMSO-d₆): 7.80 (m, 4H), 7.46 (m,3H), 6.99 (m, 1H), 5.59 (m, 1H), 5.25 (s, 2H), 4.46 (m, 3H), 4.03 (m,2H), 2.62 (m, 2H), 2.11(m, 1H), 0.94 (m, 6H).

EXAMPLE 44 p-Toluenesulfonyl-Val-Asp-fmk Step A. p-Toluenesulfonyl-Val

The title compound was prepared by a similar procedure as described inExample 1 step B in 13% yield from valine and p-toluenesulfonylchloride. ¹H NMR (DMSO-d₆): 7.90 (d, J=9.3, 1H), 7.63 (d, J=8.1, 2H),7.32 (d, J=8.1, 2H), 3.47 (m, 1H), 2.35 (s, 3H), 1.90 (m, 1H), 0.82-0.76(m, 6H).

Step B. tert-Butyl5-fluoro-3-[p-toluenesulfonyl-Valineamido]-4-hydroxypentanoate

The title compound was prepared by a similar procedure as described inExample 1 step C in 37% yield. ¹H NMR (CDCl₃): 7.73 (d, J=8.4, 2H), 7.30(d, J=7.2, 2H), 6.72-6.50 (m, 1H), 5.28-5.08 (m, 1H), 4.32-3.81 (m, 4H),3.45 (m, 1H), 2.65-2.45(m, 2H), 2.43, 2.41 (2s, 3H), 2.05 (m, 1H), 1.45,1.43 (2s, 9H), 0.88-0.79 (m, 6H).

Step C. p-Toluenesulfonyl-Val-Asp(OBu-t)-fmk

The title compound was prepared by a similar procedure as described inExample 1 step D in 92% yield. ¹H NMR (CDCl₃): 7.74-7.70 (m, 2H), 6.93(m, 1H), 7.31-7.27 (m, 2H), 7.03 (d, J=7.8, 1H), 6.96(d, J=8.1, 1H),5.26-4.61 (m, 3H), 3.55-3.47 (m, 1H), 2.98-2.48 (m, 2H), 2.11 (m, 1H),2.43, 2.41 (2s, 3H), 1.45, 1.42 (2s, 9H), 0.87-0.81 (m, 6H).

Step D. p-Toluenesulfonyl-Val-Asp-fmk

The title compound was prepared by a similar procedure as described inExample 1 step E in 31% yield. ¹H NMR (DMSO-d₆): 8.53-8.43 (m, 1H), 7.81(br s, 1H), 7.63 (d, J=7.4, 2H), 7.32 (d, J=7.4, 2H), 5.02-4.28 (m, 4H),2.18-2.40 (m, 2H), 2.34 (s, 3H), 1.79 (m, 1H), 0.77-0.74 (m, 6H).

EXAMPLE 45 p-Toluenesulfonyl-Phe-Asp-fmk

The title compound was prepared by a similar procedure as described inExample 1 in three steps from p-toluenesulfonyl-Phe and t-butyl3-amino-5-fluoro-4-hydroxypentanoate. ¹H NMR (CD₃OD): 7.61 (d, J=6.9,2H), 7.29-7.13 (m, 7H), 4.56-3.94 (m, 4H), 3.01-2.78 (m, 2H), 2.44 (s,3H), 2.51-2.37 (m, 2H).

EXAMPLE 46 Enzyme Activity

The activity of 2-chlorobenzyloxycarbonyl-Val-Asp-fmk as an inhibitor ofcaspase-3 was measured in a fluorometric enzyme assay. Enzyme activitywas measured using synthetic peptide substrates attached to afluorogenic leaving group. Cleavage of the synthetic substrate by theenzyme results in a fluorescent signal which is read in aspectrofluorometer or in a fluorometric microtiter plate reader.

12 concentrations of the testing compound ranged from 30 pM to 10 μMwere tested in the enzyme assay. The enzyme reaction was conducted inthe presence of 2 ng rCaspase 3 (purchased from PharMingen, a Bectondivision company, San Diego, Calif.), various concentrations of testingcompound, 10 μM caspase 3 substrate Ac-DEVD-AMC (SEQ ID NO:3) (purchasedfrom Quality Controlled Biochemicals, Inc. Hopkinton, Mass.) and caspasebuffer (20 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS and10% sucrose, pH 7.2) in a total volume of 100 μl. The enzyme reactionwas carried out in a 96-well plate and incubated at 37° C. for 30minutes. The plate was then read with a fluorescence plate reader (EG&GWALLAG 1420-002) using excitation filter at 355 nm/emission filter at460 nm. The data was analyzed using GraphPrism software. Otherinhibitors were tested using the same procedure and the results issummarized in Table II.

TABLE II Activity of Dipeptide as Inhibitor of Caspase-3 Caspase-3 IC₅₀Name (nM) 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk 363-Chlorobenzyloxycarbonyl-Val-Asp-fmk 36 Phenethoxycarbonyl-Val-Asp-fmk110 4-Chlorobenzyloxycarbonyl-Val-Asp-fmk 34Cyclohexylmethoxycarbonyl-Val-Asp-fmk 72 Ethoxycarbonyl-Val-Asp-fmk 58

As shown in Table II, the dipeptides are potent inhibitor of caspase-3.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

3 1 5 PRT Unknown Organism Description of Unknown Organism peptide ICEinhibitor 1 Ala Tyr Val His Asp 1 5 2 4 PRT Unknown Organism Descriptionof Unknown Organism caspase-1 inhibitor 2 Tyr Val Ala Asp 1 3 4 PRTUnknown Organism Description of Unknown Organism caspase 3 substrate 3Asp Glu Val Asp

What is claimed is:
 1. A compound having the Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R₁ isan optionally substituted alkyl or hydrogen; R₂ is hydrogen oroptionally substituted alkyl; R₃ is an alkyl, saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; X is O, S, NR₄ or (CR₄R₅)_(n), where R₄ and R₅are, at each occurrence, independently selected from the groupconsisting of hydrogen, alkyl and cycloalkyl, and n is 0; or X is NR₄,and R₃ and R₄ are taken together with the nitrogen atom to which theyare attached to form a saturated heterocyclic, partially saturatedheterocyclic or heteroaryl group, wherein said group is optionallysubstituted; or X is CR₄R₅, and R₃ and R₄ are taken together with thecarbon atom to which they are attached to form a saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or oxygen-containing heteroaryl group, whereinsaid group is optionally substituted; and Y is a residue of valine;provided that when X is O, then R₃ is not unsubstituted benzyl ort-butyl; and —X—R₃ is other than unsubstituted or substituted indolyl.2. The compound of claim 1, wherein R₁ is hydrogen, methyl, ethyl oracetoxymethyl.
 3. The compound of claim 1, wherein R₂ is hydrogen,fluoromethyl, acyloxymethyl, arylacyloxymethyl, aryloxymethyl,phosphinyloxymethyl, or aminomethyl.
 4. The compound of claim 1, whereinX is O, S or NR₄.
 5. The compound of claim 4, wherein X is O or NH. 6.The compound of claim 1, wherein R₃ is straight-chained or branched C₁₋₆alkyl.
 7. The compound of claim 1, wherein R₃ is straight-chained orbranched C₁₋₆ alkyl optionally substituted by hydroxy, carboxy, halogen,C₄-C₇ cycloalkyl, saturated or unsaturated heterocyclic group, aryl orheteroaryl.
 8. The compound of claim 1, wherein R₃ is optionallysubstituted benzyl.
 9. The compound of claim 1, wherein R₃ is optionallysubstituted pyridylmethyl.
 10. The compound of claim 1, whereinR₃—X—C(O)— is an antioxidant group.
 11. The compound of claim 10,wherein said antioxidant group is


12. The compound of claim 11, wherein said compound is


13. The compound of claim 1, wherein R₃—X—C(O)— is a fluorescent group.14. The compound of claim 13, wherein said fluorescent group is


15. The compound of claim 13, wherein said compound is selected from thegroup consisting of


16. A compound having the Formula II:

or a pharmaceutically acceptable salt or prodrug thereof wherein: R₁ isan optionally substituted alkyl or hydrogen; R₂ is hydrogen oroptionally substituted alkyl; X is O, S or NR₄; Y is a residue ofvaline; A is CR₆ or nitrogen; B is CR₇ or nitrogen; C is CR₈ ornitrogen; D is CR₉ or nitrogen; E is CR₁₀ or nitrogen; provided that notmore than three of A, B, C, D and E are nitrogen; and R₆-R₁₀independently are hydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl,C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy; or one of R₆ and R₇, or R₇and R₈, or R₈ and R₉, or R₉ and R₁₀ are taken together with the carbonatoms to which they are attached to form a carbocycle or heterocycle,selected from the group consisting of —OCH₂O—, —OCF₂O—, —(CH₂)₃—,—(CH₂)₄—, —OCH₂CH₂O—, —CH₂N(R₁₃)CH₂—, —CH₂CH₂N(R₁₃)CH₂—,—CH₂N(R₁₃)CH₂CH₂—, —N(R₁₃)—CH═CH—, —CH═CH—N(R₁₃)—, —O—CH═CH—, —CH═CH—O—,—S—CH═CH—, —CH═CH—S—, —N═CH—CH═CH—, —CH═N—CH═CH—, —CH═CH—N═CH—,—CH═CH—CH═N—, —N═CH—CH═N—, and —CH═CH—CH═CH—; wherein R₁₃ is hydrogen,alkyl or cycloalkyl; provided that when X is O, A is CR₆, B is CR₇, C isCR₈, D is CR₉ and E is CR₁₀, then at least one of the R₆-R₁₀ is nothydrogen.
 17. The compound of claim 16, wherein R₂ is hydrogen,fluoromethyl, acyloxymethyl, arylacyloxymethyl, aryloxymethyl,phosphinyloxymethyl, or aminomethyl.
 18. The compound of claim 16,wherein R₁ is hydrogen, methyl, ethyl or acetoxymethyl.
 19. The compoundof claim 16, wherein X is O, A is CR₆, B is CR₇, C is CR₈, D is CR₉, andE is CR₁₀.
 20. The compound of claim 16, wherein X is O, and one of A,B, C, D or E is nitrogen.
 21. The compound of claim 1, wherein saidcompound is selected from the group consisting of:2-Chlorobenzyloxycarbonyl-Val-Asp-fmk,3-Chlorobenzyloxycarbonyl-Val-Asp-fmk,4-Chlorobenzyloxycarbonyl-Val-Asp-fmk, Phenethoxycarbonyl-Val-Asp-fmk,Cyclohexylmethoxycarbonyl-Val-Asp-fmk, Methoxycarbonyl-Val-Asp-fmk,Ethoxycarbonyl-Val-Asp-fmk, Isopropyloxycarbonyl-Val-Asp-fmk,4-Nitrobenzyloxycarbonyl-Val-Asp-fmk,2,5-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,3,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,3,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,2,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk2,6-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,2,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,2,4-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,4-Ethylbenzyloxycarbonyl-Val-Asp-fmk,4-Bromobenzyloxycarbonyl-Val-Asp-fmk,4-Fluorobenzyloxycarbonyl-Val-Asp-fmk,Cyclopentylmethoxycarbonyl-Val-Asp-fmk,4-Trifluoromethylbenzyloxycarbonyl-Val-Asp-fmk,Benzylaminocarbonyl-Val-Asp-fmk, 3-Phenylpropyloxycarbonyl-Val-Asp-fmk,2,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,3,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,4-Morpholinecarbonyl-Val-Asp-fmk, 4-Pyridylmethoxycarbonyl-Val-Asp-fmk,2-Pyridylmethoxycarbonyl-Val-Asp-fmk,2,6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone,Isobutoxycarbonyl-Val-Asp-fmk, Propionyl-Val-Asp-fmk,Benzyl-glutaryl-Val-Asp-fmk, Glutaryl-Val-Asp-fmk,3-Fluorobenzyloxycarbonyl-Val-Asp-fmk,2-Fluorobenzyloxycarbonyl-Val-Asp-fmk,3-Methylbenzyloxycarbonyl-Val-Asp-fmk,2-Chloro-4-fluorobenzyloxycarbonyl-Val-Asp-fmk, and2-Naphthylmethoxycarbonyl-Val-Asp-fmk.
 22. A pharmaceutical composition,comprising a compound of claim 1 or 16, and a pharmaceuticallyacceptable carrier.
 23. A method of inhibiting cell death of a cell ortissue, comprising contacting said cell or tissue with an effectiveamount of a compound of claim 1 or
 16. 24. A method of treating orameliorating cell death in cardiac muscle of an animal, comprisingadministering to the animal in need of such treatment or ameliorating aneffective amount of a compound of claim 1 or
 16. 25. The method of claim24, wherein said cell death is in cardiac muscle tissue, and is due tomyocardial infarction, congestive heart failure, cardiomyopathy or viralinfection of the heart.
 26. The compound of claim 1, with the furtherproviso that when X is (CR₄R₅)_(n) and n is 0, then R₃ is a saturatedcarbocyclic, partially saturated carbocyclic, aryl, saturatedheterocyclic, partially saturated heterocyclic or oxygen-containingheteroaryl group, wherein said group is optionally substituted.
 27. Thecompound of claim 26, with the further proviso that when X is(CR₄R₅)_(n) and n is 0, then R₃ is a saturated carbocyclic, partiallysaturated carbocyclic, aryl, saturated heterocyclic or partiallysaturated heterocyclic group, wherein said group is optionallysubstituted.
 28. The compound of claim 27, with the further proviso thatwhen X is (CR₄R₅)_(n) and n is 0, then R₃ is a saturated carbocyclic,partially saturated carbocyclic or aryl group wherein said group isoptionally substituted.
 29. The compound of claim 1, wherein saidcompound is of Formula III:

wherein R₃ and R₄ are taken together with the carbon atom to which theyare attached to form a saturated carbocyclic, partially saturatedcarbocyclic, aryl, saturated heterocyclic, partially saturatedheterocyclic or heteroaryl group, wherein said group is optionallysubstituted.
 30. The compound of claim 29, wherein R₃ and R₄ are takentogether with the carbon atom to which they are attached to form asaturated carbocyclic, partially saturated carbocyclic, aryl, saturatedheterocyclic, partially saturated heterocyclic or oxygen-containingheteroaryl group, wherein said group is optionally substituted.
 31. Acompound having the Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R₁ isan optionally substituted alkyl or hydrogen; R₂ is hydrogen oroptionally substituted alkyl; R₃ is an alkyl, saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; X is CR₄R₅, where R₄ and R₅ are independentlyselected from the group consisting of hydrogen, alkyl and cycloalkyl;and Y is a residue of valine; provided that R₃—X—C(O)— is a fluorescentgroup and R₃ is other than unsubstituted or substituted indolyl.
 32. Thecompound of claim 31, wherein R₁ is hydrogen, methyl, ethyl oracetoxymethyl.
 33. The compound of claim 31, wherein R₂ is hydrogen,fluoromethyl, acyloxymethyl, arylacyloxymethyl, aryloxymethyl,phosphinyloxymethyl, or aminomethyl.
 34. The compound of claim 31,wherein R₃ is an optionally substituted oxygen-containing heteroarylgroup.
 35. The compound of claim 34, wherein R₃ is an optionallysubstituted coumarinylacetyl group.
 36. The compound of claim 35,wherein said group is


37. The compound of claim 36, wherein said compound is selected from thegroup consisting of


38. A compound selected from the group consisting of:3-(2-phenyloxyphenyl)propionyl-Val-Asp-fmk and3-(5-bromo-2-hydroxyphenyl)propionyl-Val-Asp-fmk.
 39. A pharmaceuticalcomposition, comprising a compound of claim 31 or 38 and apharmaceutically acceptable carrier.
 40. A method of inhibiting celldeath of a cell or tissue, comprising contacting said cell or tissuewith an effective amount of a compound of claim 31 or
 38. 41. A methodof treating or ameliorating cell death in cardiac muscle of an animal,comprising administering to the animal in need of such treatment orameliorating an effective amount of a compound of claim 31 or
 38. 42.The method of claim 41, wherein said cell death is in cardiac muscletissue, and is due to myocardial infarction, congestive heart failure,cardiomyopathy or viral infection of the heart.